Moselio Schaechter

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August 02, 2010

An Inactive Mine Provides Active Opportunities


Acid mine drainage in Spring Creek down-
stream from the Richmond Mine, part of
the Iron Mountain Mine Superfund Site
nine miles northwest of Redding, Calif.
Microbes inside the mine eat pyrite─fool's
gold─to produce sulfuric acid, creating the
most acidic groundwater ever measured.
Spring Creek flows eventually into the
Sacramento River. (UC─Berkeley) Source.

by Elio

“...a riddle, wrapped in a mystery,
inside an enigma...”

                                  Winston Churchill

Metagenomics is a fine tool indeed for surveying a microbial community in concert, treating both the cultured and uncultured equally. When the sample studied is rich in microbial variety, as often is the case, the pieces of genomes can be reluctant to reveal the genetic heritage of whole microbes. But there are a few particular environments that are dominated by a handful of species at most, and here this approach allows the reconstruction of complete genomes. That is the case with the acid mine drainage from mineral or coal mines. When mining ceases, all hell can break loose (anthropocentrically speaking). Microbes oxidize sulfides such as pyrite (iron sulfide) into sulfuric acid, which in turn solubilizes iron, copper, arsenic, silver, gold, and other heavy metals. Water no longer being pumped from the mine, this gemish of minerals emerges from seeps and other openings to become a highly toxic, low pH stream that eventually pollutes larger bodies of water. But for some bacteria and archaea, this is a juicy place to live and thrive.


A researcher collects samples of the pink
biofilm floating atop hot, green, acidic pools
in the Richmond Mine at Iron Mountain,
California. (Brett Baker/UC Berkeley) Source.

The most acidic water known anywhere emerges from the Richmond Mine, near Redding, in Northern California. This mine, inactive since 1963, has been studied by members of Jill Banfield’s lab at Berkeley, who have amassed an impressive amount of microbiological information. Favored for study are thick, slimy, pink microbial mats sitting on green water. Here, the dominant organism tends to a bacterium, Leptospirillum sp., sufficiently abundant that its genome could be reconstructed from metagenomic data. In addition, some unusual archaea found here turned out to be, as the authors say, enigmatic. They can be identified with fluorescent probes within biofilms that they term “blanket strips.” We asked Dr. Banfield for further enlightenment: Blanket strips is a term to describe the appearance of the biofilm. Imagine a pink fluffy blanket cut into strips. These are floating on hot water at a pH between 0.5 and 1.5 (low enough to dissolve the metal eyelets of the researchers’ boots). As an aside, she also shared with us that working underground at this site is like an extreme sport (it is extremely hot and humid). These organisms have been called ARMAN (for Archaeal Richmond Mine Acidophilic Nanoorganisms) and have been found in other environments as well, such as boreal wetlands and a New Zealand hot pool.

When it comes to the unique ARMAN attributes, one hardly knows where to begin. First, ARMANs are very small, even for environmental organisms, which tend to be on the slim side. ARMANs are 200-400 nm in diameter and able to pass through 450 nm filters. Their volume is a puny 0.009 µm3 to 0.04 µm3.


The taxonomic affiliations of the top blast hit for all proteins from each
genome to the KEGG genome database. Source.

Next, their genomes. For some of the analyses, the cells in question were concentrated by filtration. Genomes of the three strains under study have about 1 million bp and therefore are among the smallest known for free-living organisms. As expected from such parsimony, most of the DNA codes for proteins. Many genes are about 10% shorter than usual, which is also true for some obligate parasites such as the archaeon Nanoarchaeum equitans and the bacterium Neorickettsia sennetsu. So far, so good, but what is surprising is that their genomes straddle major evolutionary divides. Phylogenetically, they fit within the Euryarchaeota, yet the majority of their genes that can be assigned to COG’s (clusters of orthologous groups) are of the Crenarchaeota type. Just as surprising, as many as 21% of their genes look like they are bacterial! And some 25-38% of the genes, depending on the strain, have no homologies with anybody else. Where then do these waifs belong? Analysis of their 16S RNAs and several protein genes suggest that they fit near the root of the Euryarchaeota. But is this shoehorning them? We expect that the parentage of these odd organisms will be scrutinized further.


(A) An ARMAN cell on the right is being penetrated by a Thermoplasmatales lineage archaeon
on the left. Also shown is a virus attached to an ARMAN cell. Bar = 300 nm. (B) Details of the
penetration of the ARMAN wall by the Thermoplasmatales cell. Bar = 50 nm. Ar = ARMAN;
Tp = Thermoplasmatales lineage cell; Va = vacuole; VI = virus. Source.

Even now, mining the ARMAN genomes is a rewarding undertaking. One finds a number of characteristics shared with symbiotic or parasitic microbes, e.g., genes that are short, some that are split, and some that overlap with others. All this, plus the small genome size, suggests that ARMANs may not be able to make a living alone, relying instead on other members of their community for some resources. Indeed, looking at these organisms by cryo-EM reveals that, at least in some 3D reconstructions, ARMAN cell walls look like they are penetrated by other archaea belonging to the cell wall-less Thermoplasmatales. If so, who is parasitizing whom? Is this a way for the ARMANs to get nutrients? And yet more puzzles. To quote the authors: almost all cells contain a mysterious tubular organelle that is roughly 200 nm long and 60 nm wide. What’s this all about? Also visible are viruses attached to their surface. Lacking CRISPR defensive sequences, ARMANs may be ready prey to viral infections.

ISME fig

Slice of an ARMAN cell with the inner
membrane in orange and outer membrane
in yellow. Ribosomes are represented
with light blue spheres drawn to scale.
A tubular structure is shown in orange.
Such tubes range in diameter from 58 to
62 nm and the length is ~200 nm. Source.

One last point, perhaps not as surprising as the rest, but noteworthy nonetheless. These cells have only a handful of visible ribosomes, as few as 92 per cell, which suggests that they are not growing fast. For perspective, consider that a fairly slow-growing E. coli has some 10,000 of them! But in this special environment, perhaps one need not hurry to outcompete one's neighbors.

The ARMAN names may not last long, so fancier names have been proposed. Being uncultured, they have to bear the “Candidatus” designation, hence Ca. Micrarchaeum acidiphilum for one, Ca. Parvarchaeum acidiphilum for another. The Garden of Archaeal Wonders continues to surrender some of its secrets. And well it might, as more and more “archaeologists” are presently at work. Surely many more surprises are in stock. But it’s worth noting that now, given only a short few years, dedicated researchers can go from the discovery of novel and exciting organisms to the elucidation of many of their characteristics.

Baker BJ, Comolli LR, Dick GJ, Hauser LJ, Hyatt D, Dill BD, Land ML, Verberkmoes NC, Hettich RL, & Banfield JF (2010). Enigmatic, ultrasmall, uncultivated Archaea. Proceedings of the National Academy of Sciences of the United States of America, 107 (19), 8806-11 PMID: 20421484


Thanks, Elio: we are up to almost 70 Talmudic Questions! I probably need to print out the questions and post them.

On my office desk is a book, "The Intellectual Devotional." Supposed it helps you think about an interesting issue each day, to keep your mind cranking along.

You could *easily* create something similar for Microbial Supremacists.

"A Talmudic Question a day helps keep paradigm paralysis at bay!"

Mark, great minds.... Yes, we're thinking of that.
Many thanks,


From Elio:

Mark, yes, one of our first TQs asked if there are places on Earth where there is free water but no microbes. See
We got some lively answers, worth considering.

Brett, thanks for the reference and the interesting point regarding the vesicles.

To answer your question about the viruses Nathan. Yes, the viral population has been sequenced. There's a lot of ongoing work on this in Jill Banfield's lab.

Virus Population Dynamics and Acquired Virus Resistance in Natural Microbial Communities Anders F. Andersson, et al. Science 320, 1047 (2008)

FYI; In the first ARMAN paper (Science 2006) in the supporting materials we characterized ARMAN we also published EM images of vesicles in the community. These are significantly smaller (~50 nm) and do contain DNA, but NOT ribosomes. They are are too small for ribosomes. I guess these are what others have called nanobes, silly.

Thanks for the interest in ARMAN, there is surely a lot let to be discovered with them!

Nathan, that is a great point. Some Pseudomonas generate membrane vesicles that have DNA in them! And not just Pseudomonas, apparently. And not just DNA.

Add to it the gene transfer agent issue:

So sometimes, viruses may not contain viral DNA!

And finally, the problems with Nanoarchaeum equitans---the "universal primers" weren't universal enough!

So when any researcher observes small structures that look like viruses, even that contain nucleic acids, care must be taken...and I still wonder if, in these harsh and strange and wonderful environments, we might find remnants of *really* unusual horizontal gene transfer, just as Carl Woese suspected in the early days of evolution.

Has anybody sequenced these viruses? Do we know whether they are really viruses at all?

Elio, I presented this paper to my class last semester---it was almost *too* weird for them! I do think that, by examining the unusual, we can better understand the commonplace.

First off, any place there is a redox difference (and liquid water), we seem to have microbial life. In fact, that may be a Talmudic Question for you---"Is there any environment on Earth with liquid water that possesses no microbial life?" (you may have already asked that...).

The ARMANs are getting pretty tiny, but the final image you show reminds me that even in these tiny beasties, there is enough room for ribosomes! I'll bet it is pretty viscous in their, with the amount of DNA in the available volume.

I love this idea of how small a microbe can be---I watched the rise and fall of the "nannobacteria" concept, and am waiting to hear more about "nanobes":

The DNA staining might be apatite rather than DNA? I sure don't know.

As for the odd genomes, I look at ARMANs, and the weird tube-structures, and the archaeal cell-cell connections, and I wonder: are these relics of Carl Woese's "pre-Darwinian period," where organisms shared genes to an extent far beyond the current HGT promiscuity we observe (

The weird and the wonderful have much to teach us. Thanks for that post!

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